The development of selective chelators for toxic metals such as mercury (Hg) and cadmium (Cd) is of great importance due to their environmental and biological hazards. Current treatments for mercury and cadmium intoxication rely on chelation therapy. This work presents the synthesis and characterization of macrocyclic chelators with different pendent donor atoms for this application. These chelators exhibit strong chelation properties toward Hg²⁺ and Cd²⁺, as demonstrated by two-dimensional and multinuclear NMR spectroscopy, including 199Hg and 111Cd NMR, X-ray crystallography, and mass spectrometry. These techniques reveal the formation of stable metal-ligand complexes, with the heteroatom donors on the pendent arms directly coordinating the metal centers. In addition, potentiometric titrations were conducted to assess the pKa of the ligands and complexation constants of the metal complexes, which will further elucidate the chelator’s binding affinity and protonation behavior. Furthermore, the heavy-metal detoxification potential of these chelators was evaluated in vitro in HEK293T cells, offering a better understanding of their biological applicability. The results suggest that these chelators are promising candidates for the selective capture of Hg²⁺ and Cd²⁺ ions, with potential applications in environmental remediation and biological decorporation.